GDNF was originally purified, and characterized in the early 1990's as a neurotrophic factor supporting the survival and differentiation of midbrain dopaminergic neurons, and on the basis of the amino acid sequence of GDNF, it was possible to clone the GDNF gene (Lin et al., 1993). Neurturin (NRTN, also known as NTN) was isolated in 1996 based on its ability to promote survival of sympathetic neurons (Kotzbauer et al., 1996). Subsequently, persephin (PSPN, also known as PSP) and artemin (ARTN, also known as ART), were cloned on the basis of sequence homology (Milbrandt et al., 1998; Baloh et al., 1998). Reportedly, analysis of the Genome Database has indicated the unlikelihood of finding other functional GDNF family ligands (GFLs) (Airaksinen & Saarma, 2002).
A variety of biological actions have been ascribed to the GFLs. NRTN, PSPN, and ARTN, like GDNF, promote survival of midbrain dopaminergic neurons (Lin et al., 1993; Milbrandt et al., 1998; Baloh et al., 1998a). The survival of several other neuronal subpopulations in the CNS is supported by the GFLs, including central motorneurons (Milbrandt et al., 1998) and noradrenergic neurons (Arenas et al., 1995). GDNF, NRTN, and ARTN also support the survival of neurons in the PNS, including sympathetic, parasympathetic, sensory (Kotzbauer et al., 1996; Baloh et al., 1998), and enteric neurons (Hearn et al., 1998). In addition to their survival promoting effects, the GFLs also promotes neuronal differentiation (Lin et al., 1993; Baloh et al., 1998a; Yan et al., 2003; Paratcha et al., 2003).
As typical for secreted proteins, the four members of the GDNF-family are synthesized as inactive prepro-forms. The signal peptide is cleaved from the prepro-form of GDNF, and proGDNF is secreted. Further cleavage turns proGDNF into the 134 amino acid-long mature GDNF. Mature NRTN contains 100 amino acids, mature PSPN consists of 96 amino acids, and mature ARTN contains 113 amino acids (Kotzbauer et al., 1996; Milbrandt et al., 1998; Baloh et al., 1998). The GFLs are quite homologous, sharing from 53 to 64% sequence similarity. The identity of the protease(s) cleaving pro-GFLs to mature GFLs has yet to be determined.
The sequences of the four GFLs reveal the existence of seven conserved cysteine residues within the mature proteins. These residues are spaced in a similar manner as the seven conserved cysteine residues found in members of the transforming growth factor (TGF)-β superfamily. Hence, although they show less than 20% sequence similarity with the other members of the family, the GFLs are considered to be members of the TGF-β superfamily, constituting their own subfamily (Lin et al., 1993; Kotzbauer et al, 1996; Milbrandt et al., 1998; Baloh et al., 1998). All members of the TGF-β superfamily belong to the cysteine knot growth factor superfamily (Saarma & Sariola, 1999). The proteins in this family are characterized by being dimeric proteins containing a topological knot formed by three cysteine residues. Together with adjacent amino acids two of these cysteine residues form a covalent ring, through which the third cysteine passes.
Possible GFRα binding sites in GDNF have been investigated in two studies (Eketjäll et al., 1999; Baloh et al., 2000). The first study identified three negatively charged amino acids in finger 1 and one in finger 2, which are critical for binding of GDNF to GFRα1 (Eketjäll et al., 1999). These residues are placed in the most distal part of the fingers, as are four hydrophobic amino acids (one in finger 1, the rest in finger 2) also shown to be crucial for binding of GDNF to GFRα1. In addition, the flexible N-terminal region of GDNF is indicated to be of importance for binding to GFRα1. In contrast, the positively charged amino acids concentrated in the heel, did not appear to be involved in binding to GFRα1 (Eketjäll et al., 1999). Surprisingly, neither one of the identified residues in finger 2 nor one of the hydrophobic residues in finger 1 were required for GDNF binding in the presence of Ret since GDNF molecules mutated at these positions were still able to induce phosphorylation of Ret. Deletion of the N-terminal region did not inhibit Ret phosphorylation either. The authors therefore proposed the existence of two distinct binding sites for GDNF in GFRα1. One binding site consisting of GFRα1 receptors alone would be used for binding GDNF in the absence of Ret, while another binding site consisting of residues from GFRα1 and Ret would come into play, when GDNF interacts with a preassociated GFRα1-Ret complex (Eketjäll et al., 1999).
In the study by Baloh et al. (2000), it was found that two regions in finger 2 were important for GDNF-induced activation of Ret through GFRα1, whereas the flexible N-terminal was not required. Similar experiments showed that the corresponding two regions in NRTN and ARTN were important for the ability of these GFLs to activate Ret through GFRα2 and GFRα3, respectively. For NRTN and ARTN, regions comprising the most N-terminal part of the large α-helix were also required for Retactivation (Baloh et al., 2000).
In summary, the existence of more than one binding site in GFLs is indicated from the two studies, and although there appears to be some discrepancy about which finger is most important, the works of Eketjäll et al. (1999) and Baloh et al. (2000) underline the importance of the finger regions in binding of GDNF to its receptor complex and subsequent activation of Ret.
The understanding of the heterophilic interaction between NCAM and GDNF/GFRα1 is still very limited, but studies have indicated that both GDNF and GFRα1 bind to NCAM, and that the binding of NCAM to GDNF is greatly potentiated in the presence of GFRα1 (Paratcha et al., 2003). Furthermore, in the same study it was suggested that the GDNF-induced neurite outgrowth occurs independently of the FGFR and probably also independently of trans homophilic NCAM interactions. The GDNF-GFRα-NCAM interaction in fact seemed to interfere with homophilic NCAM interactions (Paratcha et al., 2003).